1. Field of the Invention
The present invention relates to a method and an apparatus for analyzing a three-dimensional internal structure of an internal structure of a sample such as an organism.
2. Description of the Related Art
Heretofore, as means for analyzing an internal structure of a sample such as an organism, a thin specimen is prepared from the sample to observe this specimen by a microscope or the like, or the sample is cut to observe its section by a microscope or a CCD camera. For example, in order to observe a cellular tissue, the cellular tissue is cut into thin slices by a microtome or the like, and observation is then made through a microscope.
However, such conventional means only permits a given section of the sample to be observed. Therefore, in the case of analyzing the internal structure of the sample from the sectional structures of the sample, it is necessary that many specimens should be prepared, and hence, a lot of time and a high technology are required.
On the other hand, as means for observing the internal structure of the sample by a non-destructive method without cutting the sample, an X-ray CT, a MRI (Magnetic Resonance Imaging) and a con-focal laser microscope are known. However, a resolution of the X-ray CT and the MRI is low, and there is a defect that any color information of the sample cannot be obtained. Furthermore, the con-focal laser microscope has a problem that the depth of an object to be observed depends on the transparency of the sample, and even if the transparency of the sample is high, the observable depth is limited to about 100 xcexcm, so that the sample having a size more than 1 mm cannot be observed.
In order to solve the conventional problems set forth above, the inventors of the preset invention have previously invented xe2x80x9can automated inspection apparatus comprising a sample surface cutting-out devicexe2x80x9d and have filed a patent application (Japanese examined Patent Publication No. 109384/1995). This automated inspection apparatus is disposed so as to be adjacent to a device such as a microtome for forming the slices of the sample, and it is equipped with an observation device and an analysis device. According to this kind of inspection apparatus, the sample is continuously cut to photograph its sectional images, and the internal structure of the sample is then reconstructed on the basis of the respective sectional images. Incidentally, this automated inspection apparatus is hereinafter called xe2x80x9ca three-dimensional internal structure microscopexe2x80x9d.
However, in the three-dimensional internal structure microscope, a light source is arranged above the sample to observe a reflected light. In consequence, there is a problem that, in the case that the transparency of the sample is high, the light reflected from the lower side of the section to be observed is also simultaneously observed, so that it is difficult to observe the cut section alone. That is to say, an observation performance depends on the transparency of the sample, and the observed sectional images are in a fuzzy state, so that it is difficult to observe the sample with a high precision.
Furthermore, in the case of carrying out a fluorescence observation of the sample, there is a problem that the fluorescence gradually deteriorates due to an illumination light which has penetrated into the inside of the transparent sample, so that the whole of the sample cannot be observed under the same fluorescent conditions.
The present invention has been developed to solve such problems. That is to say, an object of the present invention is to provide a method and an apparatus for analyzing a three-dimensional internal structure in which a sample can be continuously cut to continuously observe its sectional image without depending on the transparency of the sample, and even if the sample is colored with a fluorescent dye, the whole of the sample can be observed under about the same conditions, whereby the internal structure of the sample can be reconstructed with a high precision from the respective sectional images.
According to a first aspect of the present invention, there is provided a method for analyzing a three-dimensional internal structure comprising a sample extrusion step (a) for sequentially extruding a sample in a predetermined direction, a sample cutting step (b) for sequentially cutting the extruded sample, and a con-focal image pickup step (c) for focusing an illuminating light in the cut section portion to pick up the two-dimensional image of the cut section from a reflected light thereof.
Furthermore, according to a second aspect of the present invention, there is provided an apparatus for analyzing a three-dimensional internal structure comprising a sample extrusion device for sequentially extruding a sample in a predetermined direction, a sample cutting device for sequentially cutting the extruded sample, and a con-focal image pickup device for focusing an illuminating light in the cut section portions to pick up the two-dimensional image of the cut section from a reflected light thereof.
In accordance with the above-mentioned method and apparatus of the present invention, a sample can sequentially be extruded by the sample extrusion device and then sequentially cut by the sample cutting device, and the images of the sections thereof can sequentially be picked up by the con-focal image pickup device. Moreover, by scanning focal points by the con-focal image pickup device, the two-dimensional images of the whole of the cut section can be obtained, whereby the internal structure of the sample can be reconstructed from many two-dimensional images at different cut positions.
In addition, since the illuminating light is focused in the section portions cut by the con-focal image pickup device to observe the reflected light thereof, there is a less influence of the light in positions other than the focal position (e.g., the transparency of the sample). In consequence, the resolution of the two-dimensional images picked up can be enhanced to thereby reconstruct the internal structure of the sample with a high precision.
Furthermore, since the illuminating light is diffused in positions other than the focal positions and grows faint, a fluorescent dye in positions other than the focal point portion scarcely fades, even if the sample is colored with the fluorescent dye. Accordingly, the whole of the sample can be observed under about the same conditions.
In accordance with a preferred embodiment of the present invention, the illuminating light is a laser light. This constitution permits the laser light to be precisely focused in a predetermined position of the cut section portion (e.g., the surface of the section portion or a position apart as much as a certain distance from the surface), thereby enhancing the resolution of the picked-up images.
Furthermore, a data processor that operates the three-dimensional internal structure of the sample from the plurality of two-dimensional images, and a display that displays output data of the above data processor may be provided. Such a data processor permits the three-dimensional internal structure of the sample to be smoothly operated from many two-dimensional images (continuous sectional images) in different cut positions obtained by the con-focal image pickup device, and the output data thereof can be displayed by the display.
The con-focal image pickup device comprises a Nipkow""s disc having a plurality of pinholes, a micro lens disc having a plurality of micro lenses at the positions corresponding to the pinholes of the Nipkow""s disc, and a rotary device that integrally rotates the Nipkow""s disc and the micro lens disc. The thus established con-focal image pickup device gathers the laser light at respective corresponding pinholes by the plurality of micro lenses.
According to this constitution, the whole of the section in which the focal point of the laser light is severed can be scanned only by rotating the rotary device at high speed to obtain the two-dimensional images of the whole of the cut section in a short time.